A number of diseases are characterized by an exaggerated or untoward immune reactivity against harmless antigens. Such diseases include allergies, autoimmune diseases and inflammatory diseases. Normally, immune responses to harmless antigens are suppressed, a mechanism called tolerance. Tolerance to specific antigens, either exogenous or endogenous, may be induced either by mucosal or systemic exposure. Tolerance occurs because helper T-cells are deleted, paralyzed or suppressed by other T-cells, so called regulatory T-cells.
Allergies
Allergies are defined as enhanced immune reactivity to one or several harmless environmental antigens, so called allergens. In IgE-mediated allergies, the allergic individual mounts an IgE-antibody response to proteins in foodstuffs, pollens, animal dander, etc. The IgE-antibodies are produced by plasma cells developed from B-cells with specificity for a certain allergen. To become an IgE-producing plasma cell, the B-cell must receive help from a T-cell which is specific towards the same allergen. Activation of the T-cell by an allergen leads to the production of cytokines which promotes maturation of the B-cell into a plasma cell that produces IgE. The cytokines IL-4 and IL-13 are especially important in this respect. The subset of T-cells that produce such cytokines and help B-cells to become IgE-producing plasma cells, are called “Th2 cells” (Th=T helper cell). They commonly produce IL-5, a cytokine which promotes maturation of eosinophils in the bone marrow and activation of such eosinophils that arrive to the tissue where an allergic reaction takes place. Once IgE-antibodies are formed, they attach to masT-cells in the tissues, for example around blood vessels and in the respiratory and gastro-intestinal tracts. When the allergic individual is exposed to the allergen, e.g. via inhalation or ingestion, minute amounts of intact protein allergen is taken up into the circulation, reaches the masT-cells and binds to the IgE-antibodies. Hereby the masT-cell becomes activated and secretes a range of mediators that trigger the allergic reaction leading to symptoms forming disease entities such as hay fever, asthma, urticaria, atopic eczema, food allergy and allergic anaphylaxis.
Allergy is much more common in industrialized countries compared to developing countries, which also applies to autoimmune and inflammatory disorders. This has led to the speculation that exposure to microbes affords proper maturation of the developing immune system. However, it is not known which types of microbes are important for this to occur. There is an endless variety of bacteria, viruses and parasites, some of which might be important in providing the right type of stimuli to the immune system, others which may be ineffective, or even increase the risk of developing hypersensitivity or inflammation. For example, the microflora of the gastro-intestinal tract consists of several hundred species, some which are aerobic, while most are obligate anaerobes. The colonizing bacteria can be both Gram-positive and Gram-negative which each differ greatly in cell wall structure and their effects on the immune system.
Staphylococcus aureus Enterotoxins—Superantigens
Certain bacteria produce toxins, i.e. protein molecules with highly damaging potential. Most bacteria which produce toxins are pathogenic, i.e. cause disease. But toxin-producing bacteria may also reside in the normal flora of the respiratory and/or gastrointestinal tracts without causing harm. For example, newborn human infants are commonly colonized by toxin-producing Staphylococcus aureus (S. aureus) in their intestines during their first year of life without showing any symptoms from this colonization. The toxins these strains produce, e.g. S. aureus enterotoxin A, B, C or D, or TSST-1 (toxic shock syndrome toxin-1) have so called superantigen function.
Superantigens have a bifunctional binding capacity: they bind both to the major histocompatility complex II (MHC II) molecule of an antigen-presenting cell and to the T-cell receptor. Whereas a normal antigen only binds to T-cells that have specificity towards just that antigen, the “superantigen” binds to all T-cells that share one certain β-chain in their receptor, i.e. belongs to a certain Vβ-family. This means that they bind to and activate a large proportion (10-30%) of the T-cells in human beings or animals, resulting in a massive cytokine production that may lead to shock and severe symptoms, even death. This is the mechanism behind toxic shock syndrome caused by superabsorbent tampons. TSST-1 producing S. aureus may colonize the tampon and produce TSST-1 which is absorbed across the vaginal epithelium and cause shock. A method to prevent the development of superantigen-induced shock may be to expose mucosal surfaces to the particular superantigen prior to challenge, which leads to specific tolerance to that superantigen (but not other antigens). This desensitization has been attributed to production of IL-10 (Collins et al., Infection and Immunity, Vol. 79, No. 5, 2002).
Toxin-producing S. aureus have been implicated in the pathogenesis of eczema, because eczematous skin lesions are often colonized by S. aureus. It has been suggested that toxins elaborated by S. aureus can worsen the reaction by stimulating T-cells, leading to tissue damage.
However, this ability of superantigens to stimulate T-cells has been suggested as a therapeutic treatment of cancers, infectious and allergic diseases by the employment of the superantigen to activate specific immune responses (US 2001/046501), and in WO 2003/002143 engineered superantigens including staphylococcal enterotoxins and TSST-1 are used in treatment of various forms of cancer. In WO 1991/12818 to Lamb et al. superantigens are parenterally administered to reduce the immune response including T-cells in order to prevent or treat rejection reactions, autoimmune disease, allergic disease and harmful responses to infectious agents. The mechanism proposed is via deletion of T-cells or via induced anergy of T-cells. However a treatment that results in anergy or deletion of T-cells would not be recommended as prevention for allergy in children since decreased T-cell function would lead to a poor defense against infections.
Regulatory T-Cells (Tregs)
It is believed that allergy, autoimmune and inflammatory disorders are prevented by so called regulatory T-cells (Treg). These cells suppress activation of helper T-cells and thereby down-regulate many types of immune responses. One population of regulatory T-cells, named CD25+Treg (or CD4+CD25+CTLA-4+ T-cells), are CD4-positive T-cells that have a high density of CD25 on their cytoplasm which functions in intracellular expression. CD25+ Tregs have a capacity to down-regulate the expansion and activation of helper T-cells. Helper T-cells are T-cells which enhance immune responses such as T-cell mediated cytotoxicity, delayed type hypersensitivity and antibody production. Another marker that can be used to identify Tregs is messenger RNA for the gene Foxp3.
Tregs are produced in the thymus and exit to the periphery in the first days of life in mice. In humans, cells of the Treg phenotype are present at birth, but express lower levels of Foxp3 compared with cells from adults. It has been described that the number and function of CD25+ Treg can be increased by in vitro stimulation with polyclonal activators as well as specific antigens and transfer of these antigen-expanded cells into mice results in delayed development of autoimmune disease in susceptible mice. Repeated injection of the superantigen Staphylococcus aureus enterotoxin A (SEA) into Vβ3− and Vβ8 transgenic mice resulted in potentiated suppressive function of CD25+ Treg as well as induction of suppressive function in CD25− T-cells (T-cells that do not express CD25 on their surface and which cannot suppress helper T-cell functions). Superantigen administrated in such a way also results in an activation followed by a severe reduction in the number of T-cells in the animal (Grundstrom et al. Jour. of Immunology, 2003, 170, 5008-5017). This observed activation/reduction together with the fact that superantigen in the blood circulation leads to shock are the main reasons why Staphyloccocal enterotoxins administered into the blood is an unsuitable method of treatment.
Regulatory T-cells, so called Treg, have come into focus recently. As discussed above, Tregs have the ability to down-regulate many types of untoward immune responses, including allergy, autoimmunity and inflammatory bowel disease. Many methods have been designed to expand and activate this cell type in vitro with the purpose to transfer these expanded and activated cells back to the individual from whom they were derived.
It is previously known that activation of the human immune system by mucosal exposure to S. aureus toxins having a superantigen function, in order to expand and activate regulatory Tcells in vivo in early infancy, may be used to prevent inflammatory disorders, such as allergy. Document EP 1 789 083 B1 discloses use of a bacterial superantigen for prevention of an inflammatory disorder in newborn infants. Further, Lönnqvist et al (cf. European Journal of Immunology, 2009, vol. 39, 447-456) has shown that neonatal exposure to staphylococcal superantigen improves induction of oral tolerance in a mouse model of airway allergy.
Treatment of allergy and other inflammatory disorders in humans in general is a well studied field. However, many domestic mammals, such as dogs, cats and horses, also suffer from inflammatory disorders, such as allergy. In general, the options for alleviating inflammatory disorders in non-human mammals are very limited, due to lack of understanding of the non-human immune system.
The rising trend of allergy development is evident not only among humans but also among pet dogs. A growing number of pet dogs suffer from diseases due to lack of immunological regulation. Today allergy is one of the most common health problems for dogs, and one of the leading causes of visits to the veterinary office. It is estimated that 10-15 percent of all dogs, irrespective of breed, develop allergy. According to Agria Pet Insurance, one of the world's leading animal insurers, diagnoses having to do with allergies have increased by as much as 90 percent over the last decade and approximately 20 percent of all veterinary visits are related to allergies.
Many of the factors linked to increasing incidence of allergic disorders in humans are also consistent with the changing environment of dogs, such as decreased early infections, changes in diet, an urban environment, and other factors that decrease circulation of microbes and, hence, deprive the immune system of key stimulatory signals in early life
Two of the most common allergic disorders in pet dogs are canine atopic dermatitis (CAD) and food allergies. Canine atopic dermatitis is a pruritic skin disease with typical location and appearance, i.e. affecting the face, ears, paws, extremities, and/or ventrum. Often, the dog also has IgE antibodies to environmental allergens, but this is not clearly linked to disease presentation, also known as sensibilization. Otitis externa and skin infections due to staphylococci and yeasts commonly accompany CAD, due to impaired skin barrier defense in this disease. The typical age of onset of CAD is reported to be between 6 months and 3 years.
CAD shares many features with human atopic dermatitis, such as similar histopathology, pruritus as the predominant clinical sign and impaired skin barrier function. Just as for human allergies, no prophylactic or curative treatment is at hand for these disorders. Canine allergy is a complex, lifelong disease generally requiring lifelong treatment.
Allergen avoidance and anti-allergic drugs are the two treatment options. Since environmental antigens often cannot be avoided, the strategy of allergen avoidance is seldom effective. Symptoms may be relieved by antihistamines; local inflammation is curbed by topical steroids and, in more severe cases, T-cell activation can be dampened by cyclosporine. For many dogs with CAD, the response to pharmacotherapy is unsatisfactory. In these cases, allergen-specific immunotherapy, also known as hyposensitization, can be used. This is a practice designed for human allergic patients, administering gradually increasing quantities of and allergen extract to an allergic patient to ameliorate the symptoms associated with subsequent exposure to the allergen. At this time, there are few guidelines on when and how to use immunotherapy in dogs with CAD.
There is thus a need for improved methods to prevent inflammatory disorders, such as allergies, in domestic non-human mammals and particularly in dogs, cats, and horses.